This dissertation has been fi7—1 A microfilmed exactly as received ° BECKMAN, David Lee, 1939- SURFACTANT, PRESSURE-VOLUME CURVES, AND COMPONENTS OF LUNG COMPLIANCE IN RATS: EFFECTS OF EXPOSURE TO ONE ATMOSPHERE OF OXYGEN. The Ohio State University, Ph.D., 1967 Physiology University Microfilms, Inc., Ann Arbor, Michigan David Lee Beckman 1967 All Rights Reserved SURFACTANT, PRESSURE-VCLUME CURVES, AND COMPONENTS OF LUNG COMPLIANCE IN RATS: EFFECTS OF EXPOSURE TO CNF ATMOSPHERE OF OXYGEN DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University David Lee Seckrian, B.Sc., K. Sc The Ohio State University 1967 Approved by Advis ar Department of Fhysiology College of Medicine ACKNOWLEDGMENTS I especially wish to express my sincere appreciation to my advisor, Dr. Harold S. Weiss, who provided guidance and leadership in this project through his advice, patience and interest. The example he has shown as a scientist will be remembered far beyond the completion of this study. To Dr. Edwin P. Hiatt I owe a special debt of gratitude for the encouragement and enthusiasm which he has offered during my graduate training. He was responsible for my first interests in the area of environmental physiology and has continually given his support to this research project. Sincere appreciation is extended to Dr. Ronald A. ’.-/right, Dr. Rodney A. Rhoades and Capt. Richard Pilmer for their interest, suggest­ ions, and help in the laboratory. In addition I would like to thank Charles R. Wharton, E. Sue Xreglow and Joseph F. Pitt for their technical assistance. ii VITA Ray 11, 1939 Born - Dayton, Ohio 1962 ............ B.Sc., The Ohio State University, Columbus, Ohio 1962~1964 .... Teaching Assistant and Research Fellow, Ohio State University, Columbus, Ohio 1 9 6 4 ............M.Sc., Department of Physiology, Ohio State University, Columbus, Ohio 1965-1967 .... National Aeronautics and Space Administration Trainee PUBLICATIONS Weiss, H.S., Beckman, D.L., Wright R.A. Delayed Mortality in Adult Chickens Exposed to 1 Atmosphere Oxygen. Nature, 128:1003, 1965. Beckman, D.L. and Weiss, H.S. Pulmonary Surfactant and Fressure- volume Curves in 0^ Poisoned Rats. Federation Proc. 26-2, 196?. Beckman, D.L. and Weiss, H.S. Effects of Oxygen Toxicity on Pressure- volume Curves, Lung Compliance and Surfactant. (In preparation) Beckman, D.L. and Weiss, H.S. Effects of Small Amounts of Nitrogen on Rat Survival in High Po9. (In preparation) Beckman, D.L. and Scarpelli, D.G. Effects of Diamox on the Ultra­ structure of Rat Kidney Tubules. (In preparation) FIELDS CF STUDY Major Field: Physiology Studies in Renal Physiology. Professor Edwin P. Hiatt and Dante G. Scarpelli Studies in Environmental Physiology. Professors Harold 3. Weiss and Edwin P. Hiatt CONTENTS Page ACKNOWLEDGMENTS.......................................... ii VITA .................................................... iii FIGURES.................................................. vi T A 3 L E S .................................................. viii INTRODUCTION ............................................ 1 HISTORICAL REVIEW The Surface Lining of Lung Alveoli................. 3 Surface tension and lung mechanics............... 6 Methods for studying surfactant................. 9 The surface lining in several different species. 11 The lung lining in various pathological conditions . 1 2 Mechanical Properties of Lungs Related to Surface Tensions ........................... 15 Mechanical behavior of the lungs under nearly static conditions ............................. 15 Surface tension and pressure-volume curves........ 16 Role of tissue forces on pressure-volume curves . 18 Dynamic volume-pressure curves and compliance . 18 The Pulmonary Manifestations of Oxygen Toxicity . 19 O^rgen at one atmosphere........... ............ 19 METHODS General Procedure ................................. 22 A n i m a l s .......................................... 23 Oxygen E x p o s u r e .................................. 23 Removal of Rat Lungs ........ ........ .... 25 Pressure-volume Apparatus; Air I n f u s i o n............ 26 Saline I n f u s i o n .................................. 31 Residual Air in L u n g s ............................. 31 Perfusion of Lungs to Remove Surfactant............ 33 Pressure-volume Curves . 37 iv CONTENTS (Continued) Page Rate of Filling; Maximum Pressures; Volumes.............. 37 Perfusate Surfactant Measurements....................... 41 Lecithin Extraction Procedure................... 41 Thin Layer Chromatography - Separation of Lecithin . 43 Statistical Analysis of D a t a ............................ 50 RESULTS..................................................... 52 Oxygen Poisoned Rats ............................ 62 Comparison of Normal and Oxygen Poisoned R a t s .......... 70 DISCUSSION General .............................................. 34 Effects of Oxygen Toxicity on Pulmonary Surfactant . 85 Effects of Oxygen Toxicity on Tissue Compliance ..... 91 Lung Lecithin Values as an Index to Surface Activity. 91 Methods of Extraction of Surfactant ............ 93 Effect of Edema on Air and Saline Pressure- volume Curves....................................... 96 Oxygen Toxicity and Atelectasis - Lung Residual Volumes ........................................... 99 Static and Dynamic Compliance Measurements ....... 100 Method of Lipid A n a l y s i s ............................. 100 Saline Inflation and Deflation Curves................... 101 SUMMARY....................................................... 103 BIBLIOGRAPHY................................................. 105 v 1 FIGURES Figure Page 1. Oxygen Chamber............. 24 2. Air-Filled L u n g ................. 27 3. P-V Apparatus................................... 28 4. Block Diagram of Apparatus for Automatic Regis­ tration of Pressure Volume Curves from Excised Rat Lungs ................. ....... 29 5. Saline Filled L u n g ................................. 32 6. Perfusion Apparatus ............................... 35 7. Block Diagram of Apparatus for Collection of Pulmonary Surfactant via Perfusion of Pulmonary Circulation ........................... 3& 8. X-Y Recorder....................................... 39 9. Six Successive Air P-V Curves on an Excised Rat L u n g ....................... 53 10. Air P-V Curve from a Control Rat Before and After Perfusion to Remove Surfactant - Curves 5 and 6 Shown......... 54 11. Summary - Control Pressure - Volume Curve Before and After Perfusion ....................... 55 12. Complete Sequence of P-V Curves from a Control Rat . 58 13. Control Air and Saline P-V C u r v e s .................. 60 14. Control Air and Saline P-V C u r v e s .................. 61 15. Air P-V Curve from an Oxygen Exposed R a t ............ 64 16. Summary - Oxygen Pressure - Volume Curve Before and After Perfusion....................... 66 vi FIGURES (Continued) Figure Page 17. Complete Sequence of P-V Curves - Oxygen Exposed R a t ................................... 69 18. Before Perfusion Air Curves.......................... 71 19. After Perfusion Air C u r v e s ......................... 72 20. Surface Compliance - Lecithin .............. ..... 82 vii TABLES Table Page 1. Statistical Analysis of Control P-V Curves............ 57 2. Control Saline pressure-Volume Curves ................ 63 3. Statistical Analysis of Oxygen P-V Curves .......... 67 k. Oxygen Saline Pressure-Volume Curves .................. 68 5. Statistical Analysis of Air Curves - Comparison of Control and Oxygen Exposed Rats, Complete Sequence ......... 73 6. Statistical Analysis of Air Curves - Comparison of Control and Oxygen Rats, All R a t s ......... 7^ 7. Statistical Analysis - Control and Oxygen Comparison of Paired Differences, 12 R a t s ................................... 75 8. Statistical Analysis of Saline Curves ................ 77 9. Surface Compliance Values ............................ 78 10. Comparisons Between Control and Oxygen Exposed Rats of Body Weight, Lung Weight, Buoyancy, and Lecithin ........................... 80 viii INTRODUCTION Oxygen poisoning has been studied extensively (see reviews by Bean, 1945; Roth, 1964) but no completely satisfactory explanation appears available for the observed effects on either lungs or other tissues. During exposure to one atmosphere of oxygen, respiratory distress is frequently seen, and on post mortem atelectasis, edema and alveolar thickening is frequently reported, suggesting that the lungs may be a primary target. Oxygen toxicity increases lung "stiffness" (Bean, 1945), hut it has not been shown if this change is due to increased tissue rigidity, increased surface tension or both. Increases in surface tension via loss of surfactant would appear to be a likely mechanism for atelactasis and edema at least, but the data is controversial (Pattle, 1965; Fujiwara et al.. 1964; Kennedy, 1966; Bondurant and Smith, 1962; Giammona, 1965; Collier et al.. 1965). Some evidence indicates that there are species differences. Giammona, Kemer and Bondurant (1965) for example, reported that oxygen toxicity resulted in high lung surface tension in cats and rabbits but not in rats. Fujiwara (1964 ) found no effect of oxygen on surfactant or surface tension in guinea pigs. However, it has also been suggested that the method of surfactant extraction may account for the variance in results. Giammona et al. (1965) and Levine and Johnson (1964) showed that the removal of surfactant by mincing